Skip to 0 minutes and 10 seconds This practical incorporates the serial dilution technique we demonstrated previously. It uses the Miles and Misra technique for estimating viable cell counts. This experiment shows the rapid increase in microbial numbers during the storage of defrosted food, the role of microbes in food spoilage and the dangers of refreezing thawed food. For this practical, it is safe to use uncooked vegetables because of the types of microbes present. Raw meat should not be used as it may contain pathogens. Use a 10cm3 syringe to add 5cm3 of distilled water to test tube A containing 3 freshly defrosted peas. Using a glass rod crush and mix the peas and leave it to settle.
Skip to 1 minute and 10 seconds Repeat for test tube B which contains 3 peas left at room temperature for 24 hours. You can now use the 10 fold dilution technique to produce five solutions from A – the freshly defrosted peas, and five solutions from B – the peas left at room temperature.
Skip to 1 minute and 39 seconds Put 9 cm3 of sterile distilled water into each test tube. Label these up as tubes A1 for 1/10 dilution to A5 for 1/100,000 dilution for the freshly defrosted peas and B1 to B5 for the peas left at room temperature for 24 hours, and then add 1cm3 of the sample to the first test tube.
Skip to 2 minutes and 14 seconds Mix well and then take 1cm3 to add to the second test tube.
Skip to 2 minutes and 37 seconds Repeat as per the 10 fold dilution technique, until you get 5 dilutions of freshly defrosted peas and 5 dilutions of the peas left at room temperature.
Skip to 3 minutes and 16 seconds To grow the samples, use agar plates. Use one agar plate for A – the defrosted peas, and one agar plate for B – the peas left at room temperature. Label the agar plate ready to receive the samples. Take a sample from the lowest concentration first, tube A5, and lift the lid then drop one drop onto the surface of the agar at position A5. By dropping it as close to the agar surface as possible you avoid contamination from splashing. Put the petri dish lid back on and return any remaining solution to tube A5. If using disposable Pasteur pipettes available from school suppliers the drop size is about 0.02cm3.
Skip to 4 minutes and 9 seconds With this information you will be able to calculate the number of colonies for the whole sample.
Skip to 4 minutes and 17 seconds Using the same pipette take a sample from tube A4, the next concentration up, and repeat the inoculation process. Repeat for the other tubes. Allow the drops to be absorbed into the agar. Repeat this technique with the samples in tubes B5 to B1 made from the peas left at room temperature for 24 hours.
Skip to 4 minutes and 51 seconds Tape the lids onto the petri dishes at four points. Do not tape all round as this allows anaerobic pathogens to grow. If you have a group that you cannot trust not to open the dish you can seal the dishes all the way round just before the start of the lesson. Incubate 20-250c or at room temperature for 2 days. View the incubated plates using the Miles and Misra technique. Drops that contain large numbers of viable cells gives rise to circular areas of bacterial colonies which are not discrete. If a drop contains less than 15 viable cells it will produce a small countable number of colonies.
Skip to 5 minutes and 41 seconds You can see from these plates the difference in the number of colonies which have grown between the two pea samples and how quickly the bacterial growth increases. This can lead to discussions about risks of re-freezing frozen foods and how freezing preserves food.
This video is an example of practical work that has lots of opportunity for development. In this experiment we show you a practical exploring the rapid increase in microbial numbers during the storage of defrosted food, the role of microbes in food spoilage and the dangers of refreezing thawed food.
As you watch this video, think about where you would take a moment to involve your students and questions you might ask them to elaborate the context.
You could also use this video as another opportunity to consider where you would focus your learning outcomes and how the practical would fit within a progression plan. Post your suggestions below.